The present invention finds a particularly advantageous, but non-limiting, application in determining an initial quantity of nucleic acids in a sample subjected to a polymerase chain reaction (PCR) in real time. A technique of this type, known as “PCR quantification”, is used in particular for evaluating the number of copies of pathogenic agents (e.g. of the human immunodeficiency virus (HIV)) in a sample of body fluids taken from a patient, typically in the context of a medical checkup.
Reference is made to FIG. 1 for a brief description of the diagrammatic appearance of a real time PCR amplification curve with PCR cycle index numbers plotted along the abscissa and, in the example shown, with quantities of fluorescence emitted (in arbitrary units) as measured for each PCR cycle plotted up the ordinate. For each PCR cycle, it should be understood that the sample is subjected to temperature variations enabling DNA polymerase to amplify nucleic acids and enabling the corresponding PCR products to be detected by fluorescent molecules. By plotting the measured fluorescence Fn as a function of PCR cycle number n, variation is obtained of the type shown in FIG. 1, and comprises at least:                a first portion BN where fluorescence measurements coincide substantially with the background noise of the apparatus for measuring fluorescence;        a second portion EXP in which the measured quantities of fluorescence increase in substantially exponential manner;        a third portion LIN in which the increase in the measured quantities of fluorescence is significantly attenuated and behaves overall in substantially linear manner; and        a fourth portion PLA in which fluorescence measurements reach a plateau stage.        
It should be observed that for the initial PCR cycles (first and second portions), the population of interest increases in substantially exponential manner, whereas for the following cycles (third and fourth portions), other phenomena come into competition with growth in the population of interest, so that said growth is then damped up to the plateau stage PLA.
The document “Mathematics of quantitative kinetic PCR and the application of standard curves” by R. G. Rutledge and C. Côté, published in Nucleic Acids Research, 2003, Vol. 31, No. 16, discloses a method of estimating the unknown initial quantity of nucleic acids in a sample of interest by means of PCR. That method consists in using a plurality of samples having known initial quantities of nucleic acids, referred to as “standards”, in order to determine by interpolation the initial quantity of nucleic acids present in the sample of interest.
In general, the greater the initial quantity of nucleic acids in a sample, the sooner a detectable quantity of PCR product is obtained, i.e. the sooner a detectable quantity of emitted fluorescence is obtained. With reference to FIG. 2, relating to the prior art, it will be understood that the initial population in the standard St1 is greater than that in the standard St2 which is greater than that in the standard St3, etc., since the cycle Ct1 for the standard St1 occurs before the corresponding cycle Ct2 for the standard St2, which occurs before the cycle Ct3 for the standard St3, etc.
Thus, such a Ct cycle, corresponding to the cycle at which the fluorescence measurements reach a fluorescence threshold THR (as shown in FIG. 2), sets at an arbitrary level (typically below the background noise), and acts as a parameter representative of the initial size N0 of a population of nucleic acids subjected to the PCR cycles. Use has been made of this observation in the above-cited prior art to establish a relationship of the kind shown in FIG. 3 between cycle numbers Ct1, Ct2, Ct3, Ct4 for a plurality of standards having known initial populations, and their initial populations N01, N02, N03, N04. Thus, by plotting the cycles Ct1, Ct2, Ct3, Ct4, etc. up the ordinates and the logarithm of the initial population sizes N01, N02, N03, N04 along the abscissa, a regression slope REG is obtained. On this regression slope PEG, the cycle Ctint detected for the sample of interest is plotted (dashed-line arrow F1). By interpolation on the regression slope REG (dashed-line arrow F2), the initial population size N0int is then determined for the sample of interest.
Although that method is in widespread use, it nevertheless presents some drawbacks.
Firstly, it requires the use of a plurality of standard samples having respective known initial populations.
Secondly, the method depends on the judgment of the user, since the fluorescence threshold value, as selected by the user, has a direct influence on the values of the Ct cycles in the amplification curves, and consequently on the estimated values for the initial population size in the sample of interest. The threshold value also has an impact on the accuracy of the result, since accuracy is generally better if the threshold is selected to lie in the exponential growth stage EXP of the amplification curve. Nevertheless, in practice, it is difficult for the user to know whether the fluorescence threshold level THR that has been set does indeed correspond to the exponential stage of the curves, and does so for all of the samples (the standard samples and the sample of interest).
Finally, the method assumes without any verification that the population has the same amplification yield in the sample of interest and in all of the standard samples. Thus, if the sample of interest contains PCR inhibitors, as is typically the case, then its result will be falsely lowered.
It should thus be understood that the prior art technique depends on the fluorescence threshold THR as defined by the user. The value selected has an influence on the values of the Ct cycles and consequently on determining the initial quantity in the sample of interest. That is one of the reasons why a large amount of work has recently been undertaken to automate Ct cycle detection and make it reliable.